This proposal is directed toward development of a fluorescent spectral imaging system for simultaneous high resolution sub-cellular microscopy of multiple fluorescence probes in living cells. Recent developments in fluorescent probes, imaging instrumentation and micro- fabrication now permit building for an Image Slicing Spectrometer (ISS) for real time quantitative spectral imaging. We propose to combine our expertise in microscopy, optical design, fabrication, and imaging with newly available large format CCD cameras and fabrication techniques to develop an ISS system. ISS is a widefield method that is capable of acquiring full spectral information simultaneously from every pixel. This approach works by spatially redirecting image zones to obtain space between image lines. Next, by using a diffractive element, ISS obtains wavelength spread on the CCD camera (for more details on the system principle see Sections C and D). In this way, we unambiguously map x,y, ?;data onto the 2-D image sensor. The specific aims of the project are: (1) to construct the ISS with an initial wavelength range of 450 to 700 nm and (2) to test the Image Slicing Spectrometer against currently available spectral imaging systems in several live cell imaging applications. Work on development of imaging spectrometers for cellular imaging has thus far been hampered either by small fields of view, limited temporal-spatial-spectral resolution, requirement of extensive computations, or limited light efficiency. The Image Slicing Spectrometer proposed here is based on a concept borrowed from the astronomy field, and addresses the major difficulties previously connected with construction of a snapshot spectrometer. The Image Slicer transforms a rectangular Field of View (FoV) into a series of mini "slits", and rearranges them to create sufficient area for spectral spread and acquisition in the snapshot mode. No complicated processing is necessary and only simple remapping is sufficient to obtain a complete x,y, ?;data cube. The core of the system will be a custom-made redirecting mirror fabricated with diamond turning technology. The instrument will employ a Hamamatsu CCD camera with 4000 x 2624 pixel elements, Peltier cooling, and low-noise readout (C4742-98-24HR). Using this large format CCD, the final image data cube will be 400 x 260 x 50 (X, Y, ?) with a spectral resolution of 5 nm and ~0.5 <[unreadable]m spatial resolution. Once the system is built and optimized we will quantitatively evaluate the results from the ISS against the Zeiss LSM510 META, and an Optical Insights Spectral DV system. These evaluations will utilize "standard" fluorophore combinations, starting with two-color pairs, but including more challenging combinations such as CFP/GFP/YFP/Fluo-4, and mCherry/SNARF-1/Fura-Red. In summary the ISS has the potential to significantly advance a wide range of applications in area of cellular imaging. To further its impact, we plan to combine the ISS with optical sectioning in the future, using structured illumination, Nipkow disk confocal, and/or spatial deconvolution. Although it is beyond the scope of the present application, a 4-dimensional imaging system (X, Y, ?,;) would further improve the S/N of the data, as well as speed of 4-D imaging. The project targets the development of a modern spectrometer called Image Slicing Spectrometer enabling high resolution spectral imaging in real time. In consequence researchers will be able to rapidly advance the investigation of live cells with multiple fluorescent contrasts. The instrument's principle allows obtaining spectral information for entire image without scanning and thus improve signal to noise ratio. It also allows also efficient investigation of transient biological events. Technologies applied in the project and their low cost may potentially allow access of larger group of scientists to spectral imaging instrumentation.